"
Team:ZJU-China/n p 11.htm
From 2012.igem.org
11 Ligation:
11.1 Use fermentas T4 DNA Ligase. Follow the manufacturer’s protocols.
11.2 Set up the following reaction in a microcentrifuge tube on ice.
| Linear vector DNA | 20 - 100ng |
| Insert DNA | 10:1 molar ratio over vector |
| 10*T4 DNA Ligase Buffer | 2 μl |
| T4 DNA Ligase | 1 μl |
| Nuclease-free water | To 20 μl |
| Total volume | 20 μl |
11.3 Incubate 30 min at 22℃.
11.4 Heat inactivation of T4 DNA ligase at 65℃ for 10 min. (not necessary)
11.5 Use 10 μl of the mixture for transformation of 100μl competent cell.
